Genomic DNA extraction and genotyping
Genomic DNA was extracted from mouse tail tips using G-NTK lysis buffer
(Malumbres, Mangues, Ferrer, Lu, &
Pellicer, 1997) and proteinase K (1mg/ml) (Beijing Solarbio Science &
Technology Co., Ltd., Beijing, China) at 55o C
overnight. Proteinase K was deactivated at 95°C for 15min and PCR was
performed in 25μl reaction volume with diluted tail DNA and genotyping
primers (supplementary table 2). PCR master mix was as follow: 1.2μl of
each primer (10μM), 16.4μl of ddH2O, 1.5μl of 25mM
MgCl2, 2.5μl of 10X PCR buffer, 0.5μl of 10mM dNTP Mix
and 0.25μl of Taq DNA Polymerase. The PCR conditions were as follows:
95ºC for 5 min, 32 cycles of 95ºC 30sec, 58ºC 30sec and 72ºC 30sec, and
72ºC 10min using PCR machine by Bio-Rad, Hercules, CA, USA.